The lymphokine interleukin (IL)-15 is essential for the development, survival, and proliferation of NK cells. A peculiar feature of stimulation by IL-15 is that expression of the alpha chain of the IL-15 receptor (IL-15Ralpha) is not required on NK cells but on other cells, such as dendritic cells, that trans-present IL-15 to the low affinity IL-15 receptor (IL-15R beta-gamma) on NK cells. Trans-presentation of IL-15 in the context of a cell to cell contact raises the possibility of regulation by other receptor ligand interactions between the responding NK cell and the presenting cell. It is possible that proper stimulation of NK cells by IL-15 bound to the IL-15Ralpha requires additional signals from other receptors. Likewise, IL-15 trans-presentation could be inhibited by contact of NK cell inhibitory receptors with MHC class I on the presenting cell. These are important questions given the essential role of IL-15 in several facets of NK cell biology. To address these questions, the IL-15Ralpha chain has been expressed on a fruit fly Drosophila cell line, called SC2. The use of a cell from an evolutionary distant organism, such as the insect Drosophila, has the advantage of reducing the complexity due to the many receptor ligand interactions between human NK cells and mammalian target cells. Specific ligands of individual NK cell receptors can be expressed on SC2 cells and tested for their ability to induce signals and functional responses in NK cells. Expression of IL-15Ralpha on insect SC2 cells was sufficient to bind IL-15 and to trans-present it to NK cells. Therefore, stimulation of NK cells by trans-presented IL-15 does not require binding of other NK cell receptors to human ligands on the presenting cell.[unreadable] [unreadable] Most receptors in the immune system are expressed at the surface of immune cells where they can sense the environment and induce appropriate responses, such as attraction to chemotactic signals, adhesion to other cells, and even attack of other cells that have been marked for elimination. However, a few receptors have a transient residency in endosomes (e.g. growth factor receptors, toll-like receptors) from where they can transmit signals to the cell. Signaling from endosomes is emerging as a mechanism by which selected receptors provide sustained signals distinct from those generated at the plasma membrane. A receptor, called KIR2DL4, of the killer cell Ig-like receptor (KIR) family has the unique property of residing almost exclusively in endosomes. Unlike other KIRs that mediate either inhibition or activation of NK cell cytotoxicity, KIR2DL4 triggers the production of a limited set of cytokines and chemokines, and a few other molecules. The profile of genes activated by KIR2DL4 carries a typical pro-inflammatory and pro-angiogenic signature. The biological context in which KIR2DL4 may provide a useful response is early pregnancy, where the ligand of KIR2DL4, namely HLA-G, is produced. The non-classical major histocompatibility class I molecule HLA-G is expressed by trophoblast cells from the fetus that invade the maternal uterus. Uterine tissue in early human pregnancy is characterized by extensive vascular remodeling, invasion of fetal trophoblast cells, and by an abundance of maternal NK cells. How NKtrophoblast cell interactions influence vascular remodeling is unknown. We have shown that cytokine secretion by resting human NK cells is induced by soluble, but not solid-phase antibodies to KIR2DL4. Soluble HLA-G was endocytosed into KIR2DL4containing compartments in NK cells and in 293T cells transfected with KIR2DL4. Chemokine secretion induced by KIR2DL4 transfection into 293T cells occurred only with recombinant forms of KIR2DL4 that trafficked to endosomes. The profile of genes upregulated by KIR2DL4 engagement on resting NK cells revealed a pro-inflammatory/pro-angiogenic response. Soluble HLA-G induced secretion of a similar set of cytokines and chemokines. This unique stimulation of resting NK cells by soluble HLA-G, which is endocytosed by KIR2DL4, implies that NK cells may provide useful functions at sites of HLA-G expression, such as promotion of vascularization in maternal decidua during early pregnancy. Two major questions raised by these findings are the mechanism for transport of KIR2DL4 to endosomes, and the signaling pathway triggered by KIR2DL4 once bound to a soluble ligand within endosomes. The fact that KIR2DL4 is transported to endosomes and signals for cytokine secretion in non-NK cells demonstrates that this unique activation pathway exists in different biological contexts. [unreadable] [unreadable] The gp49B gene in the mouse is closely related to inhibitory KIR genes in humans. Expression of gp49B was initially reported in mast cells and NK cells. However, expression of gp49B was later reported on activated T cells and neutrophils. To test the biological function of gp49B mice with a deletion of gp49B were generated. Gp49B-deficient mice were apparently healthy and fertile. These mice were used to test the responses of mast cells and other cells to immune stimuli. Responses to the allergen ragweed (RW) upon challenge in the lung and in the eyes were assessed in two models of allergic inflammation. Infiltration by inflammatory cells into the lung during allergic responses was under negative control by gp49B. Furthermore, an increase in conjunctival inflammation, with a predominance of eosinophils, neutrophils, and degranulated mast cells, was observed in RW-sensitized gp49B-deficient mice that had been challenged in the eye, as compared to C57BL/6 wild type (WT) controls. Finally, an increase in allergic inflammation in the lungs of mice infected with the helminth parasite Ascaris suum, which stimulates an expansion of eosinophils, and sensitized with RW was observed upon RW challenge, as compared to C57BL/6 WT controls. The observed influx of eosinophils into mucus membranes of the lungs is characteristic of allergic asthma, and may contribute to airway hyperresponsiveness (AHR), airway remodeling, and mucus production. Expression of gp49B was detected on peripheral eosinophils of control mice and on eosinophils from lungs of mice treated with RW, suggesting a role for gp49B on eosinophils in dampening allergic inflammatory responses. To further investigate the role of the inhibitory receptor gp49B, mice carrying the gp49B deletion are being bred with several other lines of mice, which have other defects in the control of either mast cells or eosinophils.